Hereditary suprabasilar acantholytic mechanobullous dermatosis in buffaloes (Bubalus bubalis).

A skin disease characterized by trauma-induced sloughing of haired skin, hooves, and horns is described in four calves from a herd of Murrah buffaloes (Bubalus bubalis) in Brazil. Affected calves were detected shortly after birth by the presence of lesions affecting the distal extremities, the scapular and gluteal regions, and the tip of the tail. On histologic evaluation of affected skin, the lesions were characterized by suprabasilar vesicles and acantholysis affecting the epidermis and outer root sheath of the hair follicle infundibulum. The basal cell layer was intact and appeared as a single layer of cuboidal cells attached to the dermis. Ultrastructurally, the region between the stratum basale and the lower stratum spinosum had widened intercellular spaces with loss of desmosomal attachments, which led to the suprabasilar separation. The disease appears to be inherited as an autosomal recessive trait.

Size and charge of the functional 1,25-dihydroxyvitamin D receptor in porcine intestine.

The 1,25-dihydroxyvitamin D3 receptor from porcine intestine has been characterized by immunoprecipitation and immunoblotting experiments. Immunoprecipitation studies demonstrate that monoclonal antibodies to the receptor that recognize two different epitopes on the receptor protein quantitatively precipitate the 1,25-dihydroxyvitamin D3 binding activity from nuclear and whole cell extracts of intestinal mucosa. These antibodies were then used to study the porcine receptor by both one- and two-dimensional immunoblotting techniques. One-dimensional immunoblots of nuclear extract and whole cell extract show the receptor to be a single polypeptide of Mr approximately 55,000. The migration of the receptor on one-dimensional gels was unchanged by preassociation in vitro with 1,25-dihydroxyvitamin D3. In two-dimensional immunoblots two receptor proteins were detected, both of molecular weight 55,000. In addition, a form of the receptor did not enter the isoelectric focusing gel from either the acidic or basic direction. It did, however, migrate in the Mr 55,000 region in the molecular separation. The major receptor protein has a pI of 6.1; the minor protein has a pI of 5.9. These studies represent the first determinations of the size and charge of a mammalian 1,25-dihydroxyvitamin D3 receptor in crude tissue preparations, and they indicate that the pig receptor is a Mr 55,000 protein that exhibits charge heterogeneity in vivo. The nature of this apparent charge heterogeneity and its significance in receptor function are under investigation.

Monoclonal antibodies to the porcine intestinal receptor for 1,25-dihydroxyvitamin D3: interaction with distinct receptor domains.

Monoclonal antibodies to different domains of the porcine intestinal 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor have been produced. A nuclear extract enriched in the 1,25-(OH)2D3 receptor was prepared from small intestinal mucosa of young pigs. The receptor was purified an additional 6600-fold by chromatography on DNA-cellulose, ammonium sulfate precipitation, gel filtration high-performance liquid chromatography, and DEAE-Sepharose chromatography, with an overall yield of 23% and an average purity of 24%. A BALB/c mouse immunized with this material developed serum polyclonal antibodies to the 1,25-(OH)2D3 receptor, as demonstrated by a change in sedimentation of the porcine receptor on sucrose gradients. Spleen cells from this animal were fused with mouse myeloma cells (P3-NSI/1-Ag4-1, SP2/0-Ag14), and 24 hybridomas secreting antibodies to the 1,25-(OH)2D3 receptor were identified by both a radiometric immunosorbent assay and an immunoprecipitation assay. Twenty-one hybridoma lines were cloned by limiting dilution and further characterized as subclass IgG1 antibodies with the exception of one which is an IgA. All but two of the antibodies cross-react with the 1,25-(OH)2D3 receptor from both mammalian (human, monkey, and rat) and avian (chicken) intestine; two antibodies recognize only porcine intestinal receptor. All antibodies are unreactive to the vitamin D serum transport protein. Eight of the antibodies bind denatured receptor on an immunoblot. A solid-phase competition assay was used to identify four groups of antibodies that bind to distinct epitopes on the 1,25-(OH)2D3 receptor. One antibody from each of the four groups was used to examine the effect of antibody binding on the DNA-binding activity of the receptor-hormone complex. One antibody completely inhibited the binding of the 1,25-(OH)2D3 receptor complex to DNA-cellulose, suggesting that the epitope for this antibody may be located in the polynucleotide binding domain of the protein. Antibodies from two additional groups only slightly perturbed DNA binding, while one had no effect, suggesting that these antibodies bind to receptor epitopes distant from the region of the polypeptide directly involved in polynucleotide binding. These antibodies that are directed to several different binding sites on the 1,25-(OH)2D3 receptor provide important new tools to probe the biochemistry and topology of the 1,25-(OH)2D3 receptor and to investigate its role in mediating target tissue response to hormone.

Nutritional balance studies in very-low-birth-weight infants: role of whey formula.

Although the optimal type and amount of protein for feeding very-low-birth-weight (VLBW) infants is not well defined, a protein composition with a whey protein to casein ratio of 60/40 is generally considered desirable. This study used the metabolic balance technique to compare nitrogen retention rates in 19 VLBW (less than 1,530 g) infants fed either an experimental whey protein-predominant formula (WPF) containing ultra filtered whey protein or a conventional casein-predominant formula (CPF) containing approximately 20% more protein and more minerals. Blood chemistries and anthropometric measurements were assessed serially. Although infants fed CPF received and retained more protein than infants fed WPF, nitrogen retentions were 73.1 and 74.5% of intake, respectively, and not different between the two feeding groups. The data suggest that proteins from either WPF or CPF are adequately utilized by VLBW infants. Although WPF permitted nitrogen retention rates similar to fetal accretion rates, CPF more nearly met estimated nitrogen requirements of the low-birth-weight infant. Infants fed WPF showed a more favorable course with respect to their metabolic acid-base status, characterized by normal buffer base concentrations and less predisposition to metabolic acidosis. We conclude that whey-predominant protein is preferable to casein-predominant protein in the diet of VLBW neonates because it may lessen the risk of metabolic acidosis and its potential adverse effects.

A radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies.

A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approximately 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D3 and should be useful for the detection of antibodies to ligand-binding proteins in general.

Monoclonal antibodies to human vitamin D-binding protein.

Monoclonal antibodies to vitamin D-binding protein isolated from human serum have been produced. The antibodies obtained have been shown to be specific for human vitamin D-binding protein by three independent assays. The antibodies recognize human vitamin D-binding protein specifically in an enzyme-linked immunosorbent assay. Human vitamin D-binding protein is detected specifically in both pure and crude samples by a radiometric immunosorbent assay (RISA) and by an immunoprecipitation assay. The anti-human vitamin D-binding protein antibodies cross-react with monkey and pig vitamin D-binding protein, but not with vitamin D-binding protein from rat, mouse, or chicken, as determined by the RISA and immunoprecipitation assays.

Identification of the porcine intestinal 1,25-dihydroxyvitamin D3 receptor on sodium dodecyl sulfate/polyacrylamide gels by renaturation and immunoblotting.

Identification of the porcine 1,25-dihydroxyvitamin D3 receptor protein on NaDodSO4/polyacrylamide slab gels was accomplished by two separate techniques: (i) assay of the specific binding activity of tritiated 1,25-dihydroxyvitamin D3 to protein eluted from NaDodSO4/polyacrylamide gels and renatured and (ii) immunoblotting of the partially purified receptor using two anti-receptor monoclonal antibodies. The porcine receptor preparation used in these studies was isolated from a crude nuclear extract of intestinal mucosa followed by chromatography on DNA-cellulose, ammonium sulfate precipitation, gel filtration HPLC, and DEAE-Sepharose chromatography. These receptor fractions were then electrophoresed on NaDodSO4/polyacrylamide gels. The receptor was eluted from the gel, renatured, and assayed for its ability to bind tritiated 1,25-dihydroxyvitamin D3. The renatured receptor appears as a single peak of specific tritiated 1,25-dihydroxyvitamin D3 binding activity. This binding activity corresponds to a band on a silver-stained gel that correlates with the receptor peak eluted from the DEAE-Sepharose column. It also corresponds to the highest molecular weight species identified on an immunoblot with anti-receptor monoclonal antibodies. The 1,25-dihydroxyvitamin D3 receptor protein has a molecular weight of 55,000 as deduced from its migration on NaDodSO4/polyacrylamide gels.

1,25-Dihydroxyvitamin D3 in teeth of rats and humans: receptors and nuclear localization.

Autoradiographic and biochemical studies were used to demonstrate 1,25 (OH)2 vitamin D3 target cells in teeth. Incisor pulp of rats and molar pulp of humans were incubated in vitro with 3H-1,25 (OH)2 vitamin D3. Subsequent frozen-section autoradiography revealed a large population of cells in the pulp of both incisors and molars which selectively concentrated radioactivity in their nuclei. Extracts of incisor pulp from mature rats were found to bind 3H-1,25 (OH)2 vitamin D3 and this binding was displaceable with excess 1,25 (OH)2 vitamin D3. Sucrose density analysis revealed that the protein in tooth pulp which binds 1,25 (OH)2 vitamin D3 sediments at 3.2-3.5S. The 1,25 (OH)2 vitamin D3 receptor of intestine and kidney also sediments in this region, indicating that the 1,25 (OH)2 vitamin D3 binding protein of tooth pulp is similar to that found in other target organs. These autoradiographic and biochemical data indicate that pulpal cells of mature rat and human teeth contain receptors for 1,25 (OH)2 vitamin D3.

1 alpha,25-Dihydroxyvitamin D-induced modification of a cytosolic protein in embryonic chick intestine.

Two-dimensional electrophoresis together with radiolabeling experiments was used to examine cytosolic proteins of embryonic chick duodenum for responses to 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 caused a striking decrease in [3H]leucine content of an 18,000-dalton protein (approximate pI, 5.1) after a 10-min pulse with radioisotope followed by a 4-h chase. Decreased [14C]leucine content of the same protein was also observed at various times following 1,25-dihydroxyvitamin D3 addition to culture media; a significant decrease in radiolabel incorporation occurred within 30 min after addition of the hormone. The results argue that 1,25-dihydroxyvitamin D3 causes either a decreased synthesis rate or a post-translational modification of this protein. This change joins the biosynthesis of calcium-binding protein as an early event in the response of chick embryonic intestine to 1,25-dihydroxyvitamin D3.